Hydrogen sulfide promotes stomatal development through persulfidation of EPF peptides in Arabidopsis

Hydrogen sulfide (H₂S) plays a significant role in various processes related to plant growth, development, and responses to environmental stress, particularly affecting stomatal behavior. While numerous studies have documented the impact of H₂S on stomatal movement, the effects of H₂S on stomatal development remain relatively unexplored, and the underlying mechanisms are not fully understood. Epidermal pattern factors (EPFs) are crucial initiators of the genetic regulatory network governing stomatal development. This study aims to elucidate the regulatory mechanisms of H₂S in leaf stomatal development, thereby enhancing our understanding of the role of H₂S as a gasotransmitter in plant responses to stress. Observations utilizing light microscopy and cryo-double beam scanning electron microscope revealed that both exogenous and endogenous deficiencies of H₂S led to a significant reduction in stomatal density in the newborn leaves of Arabidopsis thaliana. Additionally, the stomatal density of 35S::EPFL9 was also diminished following the exogenous removal of H₂S, indicating that H₂S may play a role in EPFs-mediated stomatal development. More precisely, EPFL9, EPF2, and EPF1 respond sequentially to H₂S signaling at the transcriptional level. Furthermore, label-switching assays indicate that EPF2 and EPFL9 can be persulfidated by H₂S at the post-translational level, suggesting that EPF2 and EPFL9 may be the primary responders in this process.